title: Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis.
abstract: Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Stefan Meier Strasse 17, 79104, Freiburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tholen
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.forename: Martina
authors.initials: M
authors.lastname: Gansz
authors.forename: Theresa D
authors.initials: TD
authors.lastname: Ahrens
authors.forename: Manuel
authors.initials: M
authors.lastname: Schlimpert
authors.forename: Jayachandran N
authors.initials: JN
authors.lastname: Kizhakkedathu
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinheckel
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002401:Cathepsin B / Q000172:deficiency* / Q000378:metabolism; D056668:Cathepsin L / Q000172:deficiency* / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D024042:Collagen Type I / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D059748:Proteolysis; D053719:Tandem Mass Spectrometry
chemical_list: D024042:Collagen Type I; D002401:Cathepsin B; D056668:Cathepsin L; D020778:Matrix Metalloproteinase 2